Coronaviruses encode six different human pathogens that target the upper and lower respiratory tract, resulting in mild to life-threatening disease. Two recently emerged human betacoronaviruses, the group 2b severe acute respiratory coronavirus (SARS-CoV) and the group i2c Human Betacoronavirus 2c EMG/2012 (HCoV SAI) emerged suddenly from bats to produce either a global outbreak or sporadic cases of severe acute respiratory distress syndrome with high mortality in human populations. While SARS-CoV is a category C select agent, both viruses encode a single-stranded positive polarity RNA genome of about 30,000 nucleotides in length, which encode for about 29 viral proteins, including several completely unique accessory ORFs that may contribute to in vivo pathogenesis. Importantly, coronaviruses encode a variety of unknown and hypothetical ORFs as well as noncoding RNAs and miRNAs. We test the hypothesis that subsets of these novel genes play critical roles in regulating virus replication efficiency and in vivo pathogenesis. To address this hypothesis, the proposal takes advantage of novel expression vector platfomis, synthetic gene design, reverse genetics, animal models of human disease, and a defined set of biochemical and immunologic assays to identify, characterize and then determine their role in SARS-CoV, EMC-1 and select ancestral bat coronaviruses in vitro and in vivo. Specifically, we test the hypothesis that these viral genes function antagonize/modulate the host intracellular milieu to enhance virus replication, most likely be altering p53, innate immune sensing, inflammasome, apoptosis orTLR signaling. In Aim 1, we characterize the expression, subcellular localization and function as an antaogonist or agonist of antiviral defense signaling networks. In Aiim 2, we isolate recombinant virus and study the function of these uncharacterized genes in virus replication and host cell antiviral defense signaling networks. In Aim 3, we study the pathogenesis of recombinant viruses lacking unknown and hypothetical ORFs and noncoding RNAs using young and aged animal models of human disease.